Mitochondrial diseases are clinically heterogeneous diseases due to defects of the mitochondrial respiratory chain and oxidative phosphorylation, the biochemical pathways that converts energy in electrons into adenosine triphosphate (ATP). The respiratory chain is comprised of four multi-subunit enzymes (complexes I-IV) that transfer electrons to generate a proton gradient across the inner membrane of mitochondria and the flow of protons through complex V drives ATP synthesis (DiMauro and Schon 2003; DiMauro and Hirano 2005). Coenzyme Q10 (CoQ10) is an essential molecule that shuttles electrons from complexes I and II to complex III. The respiratory chain is unique in eukaryotic, e.g., mammalian, cells by virtue of being controlled by two genomes, mitochondrial DNA (mntDNA) and nuclear DNA (nDNA). As a consequence, mutations in either genome can cause mitochondrial diseases. Most mitochondrial diseases affect multiple body organs and are typically fatal in childhood or early adult life. There are no proven effective treatments for mitochondrial diseases. CoQ10 and its analogs have been administered to patients with mitochondrial disease to enhance respiratory chain activity and to detoxify reactive oxygen species (ROS) that are toxic by-products of dysfunctional respiratory chain enzymes.
An important subgroup of mitochondrial diseases is mitochondrial DNA depletion syndrome (MDS), which encompasses clinically and genetically heterogeneous disorders with reduction of mitochondrial DNA (mtDNA) copy number in tissues, leading to insufficient synthesis of mitochondrial respiratory chain complexes (RC) (Hirano et al., 2001). Mutations in several nuclear genes have been identified as causes of infantile MDS, including TK2, DGUOK, POLG, POLG2, SLC25A4, MPV17, RRM2B, SUCLA2, SUCLG1, TYMP, OPA1, and C10orf2 (PEO1) (Bourdon, et al. 2007; Copeland 2008; Elpeleg, et al. 2005; Mandel, et al. 2001; Naviaux and Nguyen 2004; Ostergaard, et al. 2007; Saada, et al. 2003; Sarzi, et al. 2007; Spinazzola, et al, 2006).
One of these genes s is TK2 which encodes thymidine kinase (TK2), a mitochondrial enzyme required for the phosphorylation of the pyrimidine nucleosides (deoxythymidine and deoxycytidine) to generate deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) (Saada, et al. 2001). Thus, mutations in TK2 impair the mitochondrial nucleoside/nucleotide salvage pathways required for synthesis of deoxynucleotide triphosphate (dNTP), the building blocks for mtDNA replication and repair.
TK2 deficiency was first described in 2001 by Saada and colleagues (Saada, et al. 2001), in four affected children originating from four different families, who suffered from severe, devastating myopathy. After an uneventful early development, at ages 6-36 months the patients developed hyperCKemia, severe muscle hypotonia with subsequent loss of spontaneous activity. Depletion of mtDNA was identified in muscle tissue with 16-22% of residual mtDNA. The patients harbored recessive TK2 mutations causing severe reductions in TK2 activity. As a consequence of mtDNA depletion, enzymatic activities of complexes I, III, IV and V of the mitochondrial respiratory chain in muscle were significantly reduced, whereas the activity of complex II, the only complex that does not contain mtDNA-encoded proteins, was relatively normal. The disease was rapidly progressive and two patients were mechanically ventilated at 3 year, while two other patients were already dead by the time of the report.
After the first description, sixty additional patients have been reported in literature and at least twenty-six further patients have been diagnosed but not reported (Alston, et al. 2013; Bartesaghi, et al. 2010; Behin, et al. 2012; Blakely, et al. 2008; Carrozzo, et al. 2003; Chanprasert, et al. 2013; Collins, et al. 2009; Galbiati, et al. 2006; Gotz, et al. 2008; Leshinsky-Silver, et al. 2008; Lesko, et al. 2010; Mancuso, et al. 2002; Mancuso, et al. 2003; Marti, et al. 2010; Oskoui, et al. 2006; Paradas, et al. 2012; Roos, et al. 2014; Tulinius, et al. 2005; Tyynismaa, et al. 2012; Vilà, et al. 2003; Wang, et al. 2005). All ninety patients had proximal muscle weakness with mild to severe respiratory insufficiency and an increased creatine kinase level up to 20-fold above normal. Disease onset ranged from birth to 74 years of age, but the majority of patients had infantile (less than 1 year, 34.4%) or childhood onset (1-12 years, 46.6%) weakness. Adult-onset (18 years or older) was reported in 14.4% of patients while only 2.2% showed first disease symptoms in adolescence (12-17 years). Global motor function was severely impaired in 47/58 (81% with Karnofsky or Lansky Performance Status <50): 46 had motor regression and were wheelchair-bound at the last follow-up while one never acquired the ability to walk. Twelve patients (3 children, 9 adults; 19%) had motor function compatible with nearly normal daily life at the last follow-up. They were able to walk independently, but required support for long distance, climbing stairs, or both. Data from motor rating scale were not available for 31 patients. Respiratory muscles were severely compromised in 30/48 (62.5%) patients, who required mechanical ventilation or nocturnal/continuous non-invasive ventilation (data were not available in 42 patients). Other muscular functions were affected in 31/83 (37.3%) patients who manifested a variable combinations of: dysarthria/dysphasia (3); rigid spine (1); mild dysphagia (9); facial diplegia (19); ptosis (22); and progressive external ophthalmoparesis (PEO) (12). Sixteen patients required gastrostomy tube because of severe dysphagia and failure to thrive. Central nervous system was affected in 13 out of 90 (14.5%) causing: recurrent seizures (6); encephalopathy (5); cognitive impairment (4); coma episodes (1); and sensorineural hearing loss (3). TK2 deficiency caused death in the first 3 years of life in 50% of the patients; 24/41 (58.5%) had infantile-onset while 7/41 (17%) had a childhood onset. Only 6.25% of patients have lived more than 42 years.
Nerve conduction studies (NCSs) and electromyography (EMG), performed in 40 patients, showed: myopathic changes defined by polyphasic short duration low amplitude motor units potentials in 32/40 (80%); “myopathic and neuropathic” changes in 3/40 (7.5%); sensory axonal neuropathy by NCS in one (2.5%); chronic denervation by EMG in another patient (2.5%); low amplitude in the facial nerve in one patient (2.5%); isolated “neuropathic” changes, in 2/40 (5%); and no abnormalities in three patients (7.5%). Muscle biopsies revealed variable depletion and multiple deletions of mitochondrial DNA (mtDNA).
Based on clinical and molecular genetics findings, three disease presentations were identified: i) infantile myopathy (37.8%) with onset of weakness in the first year of life with severe mtDNA depletion and early mortality; ii) childhood SMA-like myopathy (35.6%) with severe mtDNA depletion; and iii) adult myopathy (26.7%) with mild weakness at onset and slow progression to loss of ambulation, respiratory insufficiency, or both, often with chronic progressive external ophthalmoparesis in adulthood in association with mtDNA multiple deletions, reduced mtDNA copy number, or both. Thus, TK2 deficiency manifests a wide clinical and molecular genetic spectrum with the majority of patients manifesting in early childhood with a devastating clinical course, while others have slowly progressive weakness over decades.
Histological and histochemical study showed type I fiber prevalence, atrophic fibers with lipid storage and increased connective tissue, ragged red fibers, cytochrome c oxidase (COX, complex IV) negative fibers with succinate dehydrogenase (SDH, complex II) hyperactivity. In the end stage, muscle is replaced by fatty tissue, including the respiratory muscle, as evident in the chest MRI of one patient (Collins, et al. 2009).
Recently, adult cases have been reported, four with slowly progressive myopathy and two with PEO. In these patients the levels of mtDNA depletion was not severe and was associated with multiple deletions of mtDNA (Béhin, et al. 2012; Paradas, et al. 2012; Tyynismaa, et al. 2012).
Treatment for TK2 deficiency, like most MDS and mitochondrial disorders, has been limited to supportive therapies. Thus, there is a need for better therapeutic intervention, and understanding the pathomechanism of MDS would allow the design of treatment strategies targeting either the cause of the disease or the downstream metabolic defects, making for more effective therapies.